Proteoglycans in human long-term bone marrow cultures: Biochemical and ultrastructural analyses

Abstract

Proteoglycans within the extracellular matrix of human bone marrow have been implicated in the process of hematopoiesis, but little is known about the structure and composition of these macromolecules in this tissue. Hematopoietically active human long-term bone marrow cultures were incubated with medium containing 35S-sulfate and 3H-glucosamine as labeling precursors. Proteoglycans present in the medium and cell layer were extracted with 4 mol/L guanidine HCl and purified by diethylaminoethyl (DEAE)-Sephacel ion exchange and molecular sieve chromatography. Both culture compartments contain a large chondroitin sulfate proteoglycan (MI, CI) that eluted in the void volume of a Sepharose CL-4B column and contained glycosaminoglycan chains of molecular weight (mol wt) ~38,000. A second population of sulfate-labeled material was identified as a broad heterogenous peak (MII, CII) that was included on Sepharose CL-4B at K(av) = 0.31. This material when chromatographed on Sepharose CL-6B could be further separated into a void peak (MIIa, CIIa) and an included peak eluting at K(av) = 0.39 (MIIb, CIIb). The void peaks (MIIa, CIIa) were susceptible to chondroitinase ABC digestion (99%) but slightly less susceptible to chondroitinase AC digestion (90%). Papain digestion of these peaks revealed them to be proteoglycans with glycosaminoglycan chains of mol wt ~38,000. The included peaks on Sepharose CL-6B (MIIb, CIIb) from both medium and cell layer compartments resisted digestion with papain, indicating the presence of glycosaminoglycan chains of mol wt ~38,000 either free or attached to a small peptide. Although this material was susceptible to chondroitinase ABC (98%), it was considerably less susceptible to chondrotinase AC (~60%), indicating that it contained dermatan sulfate. A small amount of heparan sulfate proteoglycan was also identified but constituted only ~10% of the total sulfated proteoglycan extracted from these cultures. Additionally, approximately 40% of the incorporated 3H-activity radioactivity was present as hyaluronic acid. Electron microscopy revealed a layer of adherent cells covered by a mat containing ruthenium red-positive granules that were connected by thin filaments. The extracellular matrix layer above the adherent cells contained a mixture of hematopoietic cells. Chondroitinase ABC treatment of the cultures completely removed the ruthenium red-positive granules overlying the cells and resulted in a loss of ~70% of the 35S-sulfate-labeled material from the cell layer. This culture system is offered as a model to investigate the role of proteoglycans in hematopoiesis.